DESCRIPTION (Applicant's Abstract): Cocaine has toxic effects on the brain and heart causing seizures, cerebral hemorrhage, a marked increased in heart rate, disturbance of heart rhythm, and hypertensive crisis. Death can occur even in young people. It is estimated that 6 million Americans are regular users of cocaine and that emergency rooms annually treat 100,000 patients for cocaine related problems. Treatment with purified human butyryl cholinesterase (EC 3.1.1.8) protected rats from cocaine-induced convulsions, lethality, cardiac arrhythmias, hypertension, and hyperactivity. Butyryl cholinesterase is the major detoxifying enzyme of the pharmacologically active cocaine isomer, (-)-cocaine. Butyryl cholinesterase hydrolyzes (-)-cocaine at a rate of 4 molecules of cocaine per molecule of enzyme per minute (kcat=4 per min). This rate is slow compared to the rate at which butyryl cholinesterase hydrolyzes the pharmacologically inactive cocaine isomer, (+)-cocaine, where kcat=10,000 per min. Our goal is to genetically engineer human butyryl cholinesterase to enable it to hydrolyze (-) cocaine at a rate approaching the rate at which it hydrolyzes (+)-cocaine. The difference between (-) and (+) cocaine is the position of a methyl ester group. It is expected that fitting this small group into the active site will require mutation of a few amino acids in the active site gorge. Mutants made with the polymerase chain reaction will be expressed in Chinese hamster ovary cells. The secreted enzymes will be purified and assayed for cocaine hydrolase activity. The enzyme with the highest kcat and highest binding affinity will be tested in rats to measure how much of the enzyme is needed to provide full protection from cocaine-induced toxicity, and to measure the efficiency with which it reverses cocaine toxicity. The product of this research, a human butyryl cholinesterase that rapidly hydrolyzes (-)-cocaine, has the potential to be useful for treating cocaine intoxicated patients. A final goal is to test the hypothesis that people who have genetic variants of butyryl cholinesterase are more susceptible to the toxic effects of cocaine. Test tube experiments have shown that the atypical variant (D70G) of butyryl cholinesterase has a 30 fold decrease in binding affinity for cocaine. This predicts that people with the atypical variant will react to a standard dose of cocaine as if it were an overdose, similar to their abnormal response to the muscle relaxant succinylcholine. To test this hypothesis, it is proposed to genotype the butyryl cholinesterase of people who have died after using cocaine. It is expected that butyryl cholinesterase genetic variants will be present in a higher frequency in cocaine-related fatalities.